Gel microdroplets in genetic analysis

ABSTRACT

The invention provides methods of nucleic acid analysis. Such methods entail forming a population of gel microdrops encapsulating a population of biological entities, each entity comprising a nucleic acid, whereby at least some microdrops in the population each encapsulate a single entity. The population of gel microdrops is then contacted with a probe under conditions whereby the probe specifically hybridizes to at least one complementary sequence in the nucleic acid in at least one gel microdrop. At least one gel microdrop is then analyzed or detected. The biological entities can be cells, viruses, nuclei and chromosomes.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application derives priority from U.S. S No.60/095,721, filed Aug. 7, 1998, which is incorporated by reference inits entirety for all purposes.

TECHNICAL FIELD

[0002] The present invention resides in the field of genetic analysis.

BACKGROUND

[0003] Cytogenetic testing is still in its infancy. Current cytogeneticmethods are limited to analysis of gene aberrations easily detectable incells, nuclei, or chromosomes, in part, because slide based methods arehighly manual. Analysis of aberrations present in low frequency is notroutinely performed in the clinical setting because many slides ofcells, nuclei or chromosomes would have to be evaluated to establishstatistical frequency.

[0004] Banding is the classical approach used for analyzing chromosomesin metaphase spreads. This method is based -on staining which results indark bands in the region of the chromosome where the chromatin occurs athigher density. The banding pattern is specific for each chromosome andallows identification for karyotyping, which is the determination ofeach chromosome's copy number. However, banding resolution is notsufficient to detect small deletions or additions of chromosomal mass,which occur in a variety of disease conditions, particularly in cancers.

[0005] Fluorescent in situ hybridization is another approach used tolocalize genomic DNA fragments or to paint whole chromosomes and todetect and characterize genetic abnormalities including translocations(31, 40), aneusomy (41, 42), and gene amplification (43). These geneticabnormalities can be detected in individual cells, chromosomes, ornuclei to assess of tumor genotype, analyze genetic heterogeneity, anddetect malignant cells. To preserve integrity in FISH applications,chromosomes are typically adsorbed onto glass slides for analysis.Analysis therefore requires microscopic evaluation of individual slideslimiting automation and rapid sample processing. Fluorescent in situhybridizations prepared on glass slides rely not only on the assay andreagents but on the instrumentation and the expertise and ingenuity ofthe scientists using it resulting in poor reproducibility. An inherentlimitation to this technology is that at least 100 kb of DNA sequence ina single cell must be present for detection (68-70). In addition, harshconditions for fixing either tissue or intact cells to a glass slide areless than optimal: up to 90% of the assay sample can be lost from theglass support.

[0006] Some chromosomes can also be resolved by fluorescent stainingfollowed by flow cytometry (14,15). Successful chromosome sorting is,however, dependent on the binding characteristics of fluorescent dyesand the extent to which the chromosome of interest can be distinguishedfrom chromosomes of similar size, clumps of chromosomes, and debriscontaining DNA (13). Although this approach has resulted in theconstruction of yeast artificial chromosome (YAC) libraries for mappingstudies (16) in species which have chromosomes of similar size, such asmouse, arabidopsis, and 20% of the human chromosomes, unambiguousresolution has not been possible. Flow sorting based on dye uptake ispossible for well resolved chromosomes, but this method works poorly forchromosomes which are similar in size and base composition, mainly humanchromosomes 9-12 and the majority of mouse chromosomes. Furthermore,flow cytometry cannot currently be used to analyze hybridizedchromosomes prepared by conventional methods because unfixed chromosomesfall apart using high temperatures and/or formamide.

SUMMARY OF THE CLAIMED INVENTION

[0007] The invention provides methods of nucleic acid analysis. Suchmethods entail forming a population of gel microdrops encapsulating apopulation of biological entities, each entity comprising a nucleicacid, whereby at least some microdrops in the population eachencapsulate a single entity. Nucleic acids can be DNA or RNA. Thepopulation of gel microdrops is then contacted with a probe underconditions whereby the probe specifically hybridizes to at least onecomplementary sequence in the nucleic acid in at least one gelmicrodrop. At least one gel microdrop is then analyzed or detected. Thebiological entities can be cells, viruses, nuclei and chromosomes.

[0008] In some methods, at -least 10,000 biological entities areencapsulated. In some methods, the biological entities are not fixedchemically before the contacting step. In some methods, nucleic acidsare amplified before the contacting step. Suitable materials for formingdroplets include agarose, alginate, carrageenan, or polyacrylamide.

[0009] In some methods, nucleic acids are recovered from microdrops bydigestion with agarase. Optionally, the recovered DNA can be digestedwith a restriction enzyme with or without prior digestion of agarase. Insome methods, the gel matrix is crosslinked with itself and/or nucleicacid being analyzed, typically, between the denaturation and contactingsteps. In some method, the hybridization is performed at a temperatureof over 68° C. or in the presence of a formamide concentration greaterthan 20%. In some methods, the microdrops further comprise a reagentthat amplifies a signal from the labelled probe. For example, the probecan be labelled with an enzyme, and the reagent can be a substrate forthe enzyme.

[0010] In some methods, microdrops are isolated by FACS™. In somemethods, the biological entities are a population of chromosomesobtained from a population of different cells in a patient. In somemethods, the ratio of a subpopulation of microdrops containing achromosome hybridized to the probe to a subpopulation of microdropscontaining a chromosome not hybridized to the probe is determined. Insome methods, the probe hybridizes to a nucleic acid segment bearing amutation and the ratio indicates the proportion of cells in thepopulation bearing the mutation. Such methods are particularly usefulfor analyzing somatic mutations.

[0011] In some methods, an isolated microdrop containing a singlechromosome is used to prepare a single chromosomal fragment library.Such a library can in turn be used for preparing probes for a singlechromosome, such as painting or reverse painting probes.

[0012] Gel microdrops encapsulated biological entities can be storedbefore or after the hybridization step for a period of at least sixmonths.

[0013] The invention further provides methods of diagnosing a diseasedue to a genetic mutation. Such methods entail obtaining a sample ofcells from a patient. A population of chromosomes from the sample inthen encapsulated in a population of microdrops. One then contacts themicrodrops with a first probe that is complementary to a nucleic acidsegment containing the somatic mutation, and a second probecomplementary to the chromosome in which the somatic mutation occurs ata site distal to the somatic mutation. The first probe hybridizes tomicrodrops bearing the chromosome with a somatic mutation and the secondprobe hybridizes to microdrops bearing the chromosome irrespectivewhether the somatic mutation is present. One then determines the ratioof microdrops hybridizing to the first probe and hybridizing to thesecond probe. The ration can then be used to diagnose the existence orprognosis of the disease from the ratio. Such methods are particularuseful for diagnosing existence or prognosis of cancer.

[0014] The invention further provides methods of chromosome analysis.Such methods entail forming a population of gel microdrops encapsulatinga population of nucleic, whereby at least some microdrops in thepopulation each encapsulate a single nucleus. One then contacts thepopulation of gel microdrops with a probe under conditions whereby theprobe specifically hybridizes to at least one complementary sequence inat least one chromosome in a nucleus of least one gel microdrop. Onethen isolates or detects the at least one gel microdrop.

[0015] The invention further provides methods of isolating chromosomes.Some such methods entail culturing a population of cells in genisteinand colcemid to synchronize chromosomes in metaphase, and isolatingchromosomes from the cells. Other methods, which can be used inconjunction or independently of the previously described methods, entaillysing a population of cells to form a lysate. The lysate is thentreated with an antibody linked to a magnetic particles, wherein theantibody specifically binds to one or more chromosomes in the cells.Magnetic particles are then isolated from the lysate.

[0016] The invention further provides methods of chromosome analysis.Such methods entail forming a population of gel micropdropsencapsulating a population of cells or nuclei, whereby at least somemicrodrops in the population each encapsulate a single nucleus. One thencontacts the population of gel microdrops with a probe under conditionswhereby the probe specifically hybridizes to at least one complementarysequence in at least one nucleus in at least one gel microdrop. One thenisolates or detects the at least one gel microdrop.

[0017] The invention further provides a kit comprising high meltingtemperature agarose, emulsification equipment, and a label indicatinghow to use the kit for probe hybridization analysis. Optionally, the kitalso includes at least one probe that hybridizes to a nucleic acid.

BRIEF DESCRIPTION OF THE FIGURES

[0018]FIG. 1 shows encapsulated and unencapsulated human, mouse andplant chromosomes.

[0019]FIG. 2 shows gel electrophoresis of chromosomal DNA.Unencapsulated, freshly isolated chromosomes: b-mouse, e-human K-562,g-human REH; Unencapsulated chromosomes one month after isolation:c-mouse, f-human K-562, h-human REH; Encapsulated chromosomes stored for6 months: d-mouse, i-human K-562, j-human REH;lambda DNA (0.1 μg) usedas a control: a,k.

[0020]FIG. 3A shows flow sorting of human chromosome 22 followingmicrodrops in situ hybridization using a bcr locus specific probe. FIG.3B shows sorted chromosomes visualized using fluorescent microscopy.

[0021]FIG. 4 shows purity of chromosome 22 after sorting measure by thepolymerase chain reaction using primer sets specific from chromosomes10, 21 or 22

[0022]FIG. 5 Restriction digestion of encapsulated chromosomes.

[0023]FIG. 6 detection of translocated chromosome 22 left chromosome 9middle, and translocation-free chromosome 22 right by MISH are shown.

[0024]FIG. 7 shows flow cytometric detection of gag HIV RNA inencapsulated HIV infected cells after hybridization with two HRP-labeledoligo probes.

[0025]FIG. 8 shows detection of telomerase mRNA in HL-60 (model cancercell line) and human PBMCs using fluorescein-labeled oligonucleotideprobes

DEFINITIONS

[0026] An isolated species means an object species invention that is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition). Preferably, anisolated species comprises at least about 50, 80 or 90 percent (on amolar basis) of all macromolecular species present. Most preferably, theobject species is purified to essential homogeneity (contaminant speciescannot be detected in the composition by conventional detectionmethods).

[0027] Polymorphism refers to the occurrence of two or more geneticallydetermined alternative sequences or alleles in a population. Apolymorphic marker or site is the locus at which divergence occurs.

DETAILED DESCRIPTION

[0028] I. General

[0029] The invention provides methods of analyzing populations ofnucleic-acid containing biological entities by probe hybridization. Forexample, the methods can be used to analyze cells, viruses, isolatednuclei or isolated chromosomes. The methods work by encapsulatingbiological entities in gel microdrops, such that at least somemicrodrops in a population contain a single entity. The gel dropletprovides a stabilization matrix for hybridization and holds hybridizednucleic acids together for subsequent analysis. Encapsulated entitiesare hybridized with one or more probes. GMDs are easily recovered usinglow speed centrifugation. Probes can, for example, be designed tohybridize to particular chromosomes or to specific chromosomal lociwhich are the site of genetic abnormality. After hybridization, theencapsulated entities are detected and/or isolated based onhybridization signal.

[0030] The methods allow very large numbers of biological entities to beanalyzed simultaneously and can detect entities with rare genotypes fromwithin such populations. For example, the methods can be used toidentify rare cancerous cells in a population of normal cells at anearly stage in the development of the cancer. Other applicationsincluded gene identification, isolating cells expressing a particulargene, preparation of specific hybridization probes, and isolation ofpure starting material for DNA sequencing. An additional benefit is thatencapsulation in a permeable matrix permits hybridization in freesolution, improving the reaction kinetics. A further benefit is that theencapsulation matrix can serve as a repository for a substrate for areaction catalyzed by an enzyme bound to a probe. Use ofchemiluminescent substrates in this manner results in highly sensitivedetection.

[0031] II. Formation of Droplets

[0032] Gel Microdrop (GMD) encapsulation evolved from an interest instudying individual cells (1-11). GMD's provide a definedmicroenvironment around a biologically entity. The gel does not impedediffusion and allows analysis of large numbers of individual GMDs usingflow cytometry, as well as recovery of GMDs of interest using FACS. Thenumber of biological entities encapsulated within each GMD isapproximated by Poisson statistics, similar to limiting dilution cloningor petri dish inoculation. To obtain a preparation with a highprobability that each GMD contains 0 or 1 initial chromosomes, about 10%of the GMDs should be occupied. GMDs can be prepared by dispersingentities in liquefied gel, such as agarose, into an excess of ahydrophobic fluid to form an emulsion. The emulsion is transientlycooled, causing gelling. Once formed, GMDs are physically distinct androbust and can be removed from the oil into an aqueous medium by lowspeed centrifugation. Alternatively, GMD's can be formed by passing amixture of liquefied gel and entities through a pulsating nozzle, suchas the printhead of an inkjet printer.

[0033] Instrumentation for microdrop formation, the CellSys 100™Microdrop Maker, is a specially designed emulsifier coupled to a highprecision motor available from OneCell Systems, Inc. By varying therotation speed, type and amount of surfactant, and emulsion viscosity,microdrops ranging from, for example, 2-200 μm can be prepared. Althoughthe Microdrop Maker currently available from One Cell Systems is mostefficient for making large numbers of microdrops (e.g., 10⁷), which inturn requires one million biological entities to meet the singleoccupancy requirement of the microencapsulation procedure, it can beminiaturized for encapsulation of smaller chromosomal preparations. Suchis useful for clinical applications, e.g., evaluation of bone marrowsamples in which only a small number of cells are present.

[0034] Several types of gel can be used for making droplets includingagarose, alginate, carrageenan, or polyacrylamide. High meltingtemperature agarose is preferred for encapsulating larger human andplant chromosomes.

[0035] III. Biological Entities

[0036] The methods are generally applicable for screening nucleic acids,and any biological entity containing them. Examples of biologicalentities include cells, organelles, such as nuclei, mitochondria andchloroplasts; chromosomes and fragments thereof, and viruses. Suchentities can be from any species including mammals, fish, amphibians,avians, insects, bacteria, eubacteria and plants. Preferred mammalsinclude humans, primates, bovines, and rodents, such as mice, rats andrabbits.

[0037] In some methods, biological entities are obtained from a tissuefrom a human patient. The tissue sample often contains a nonclonalpopulation of cells. Samples can be obtained from any tissue, but bloodsamples, and samples from tissues from the loci of diseases to which thepatient is suspected of being susceptible are preferred. In somemethods, cells from primary tissue samples are propagated beforeanalysis. In some methods, biological entities are pooled from more thanone individual before analysis. In some methods, biological entities(e.g., chromosomes) are obtained from a homogenous cell line. Cells canbe encapsulated and analyzed directly, or nuclei or chromosomes can beisolated from cells for analysis.

[0038] IV. Pretreatment of GMD's before Hybridization

[0039] Preferably, polymers forming the gel are crosslinked to eachother and/or to the biological entity. Preferably, such crosslinking isreversible without damage to the biological entity, thereby allowing thebiological entity to be recovered after hybridization and subjected tofurther DNA manipulations. Such crosslinking assists in preservation ofstructural integrity of GMD's in the subsequent denaturation step andhybridization steps. Harsh chemical fixation treatments, such as theformaldehyde treatment, used in conventional FISH, are not required.Such fixation treatments form an internal matrix by cross-linkingendogenous primary amino groups in a biological entity.

[0040] Crosslinked gel microdrops can withstand high temperatures (atleast 68°) or concentrations of denaturing solvents such as formamide(e.g., 10, 20, 30, 40 or up to 50% concentration of formamide).Optionally, segments of DNA or RNA can be amplified within the dropletsusing PCR. A PCR buffer including primers is diffused into droplets, andthe droplets are subject to temperature cycling as in a conventional PCRreaction. Irrespective whether amplification is performed, nucleic acidswithin the GMD's are typically denatured (e.g., by treatment withalkali, heat, formamide or other chemical denaturant) before performingthe hybridization step.

[0041] V. Probes

[0042] Probes are designed to hybridize with selected segment(s) in thenucleic acids of biological entities being screened. Typical probes arethose used in conventional genetic and cytogenetic analyses. In manymethods, two or more probes with different binding specifies are used.In some methods, a large population of different probes is used.Typically, probes bear detectable labels. If more than-one type of probeis used, the different types sometimes bear different labels.

[0043] Some probes used in the methods are locus-specific probes,including allele-specific probes and species-specific probes.Allele-specific probes hybridize to one allele of a gene within aspecies without hybridizing to other alleles. Similarly,species-specific probes hybridize to a gene from one species withouthybridizing to the cognate gene in another species. Some probes used inthe method hybridize to a variant form of chromosome associated withdisease without hybridizing to a wildtype form of the chromosome foundin normal individuals. Some probes are mixtures of probes designed tohybridize to one chromosome from an individual or species withouthybridizing to other chromosomes. For example, a population of probescan be designed to hybridize to the human X chromosome withouthybridizing to other human chromosomes. Some probes are mixturesdesigned to hybridize to several different chromosomes. For example, amixture of probes can be designed to hybridize to each of the humanchromosomes. Some probes hybridize to satellite or repeat regions withinchromosomes. Some probes hybridize to centromeric regions ofchromosomes.

[0044] Some probes are chromosome painting probes or reverse chromosomepainting probes. Chromosome painting probes are a collection of probesdesigned to hybridize to a segment of a chromosome. Microscopic analysisof a chromosome hybridized to such probes shows a contiguous segment oflabel if the entire segment of the chromosome is present. If the segmentis interrupted by a substitution, deletion or insertion, a gap appearsin the pattern of label, signifying the presence of a geneticabnormality. Reverse chromosome painting probes are designed tohybridize to a contiguous segment of a chromosome bearing a knownmutation. Microscopic analysis of a chromosome bearing such a mutationhybridized to reverse painting probes shows a contiguous segment oflabel.

[0045] Reverse chromosome painting has been useful for determining theorigin of de novo unbalanced chromosome duplications and the extent ofdeletions or balanced translocations (20). However, aberrant chromosomesare often difficult to distinguish in conventional FISH methods becausethe derivative chromosome can overlay normal chromosomes. Use of reversechromosome painting after separation of chromosomes by flow cytometryeliminates this problem.

[0046] Some probes are designed to bind to mRNA within a cell. Suchprobes can be designed incorporating a segment from the antisense strandof a cDNA sequence.

[0047] Probes are typically nucleic acids, and can be RNA, DNA or PNA.Probes can also be antibodies or other proteins with capacity to bind toDNA in a sequence-specific manner.

[0048] VI. Labels

[0049] Probes are typically labelled. The labels used permit separationbased on flow cytometry and/or microscopic visualization of label. Insome methods, probes are labelled with fluorescent label such asfluorescein. If multiple probes are used simultaneously the probes canbe labelled with different fluorescent molecules emitting at differentwavelengths to allow differential detection.

[0050] In some method, the signal from a label attached to a probe isamplified by binding molecules bearing secondary labels to the label.For example, a hybridized probe labelled with fluorescein can beincubated 15-30 min with rabbit anti-fluorescein IgG conjugated withbiotin (Accurate Chemical & Scientific). After washing with PBS buffer,GMDs are incubated for 15-30 min with avidin-FITC oravidin-phycoerythrin (Sigma, St. Louis, Mo.). Because, on average, eachanti-fluorescein is labeled with five biotin molecules and each biotinmolecule can bind 2-4 avidin molecules, a 10-20 fold amplification insignal is obtained.

[0051] In some methods, probes are labelled with an enzyme thatcatalyzes conversion of a substrate to a secondary label that allowsseparation and/or visualization of GMD's.

[0052] In some methods, as well as being hybridized to sequence-specificprobes, GMD's are labelled with compounds that binds to any DNAsequence. Such labelling serves to distinguish GMD's containing abiological entity with empty GMD's.

[0053] VII. Separation and Analysis of Hybridized GMDs

[0054] After hybridization with labelled probes, GMDs can be analyzed ona flow cytometer. In the simplest case, in which a single probe type isused, the flow cytometer counts the number of GMDs bearing a label andthe number of GMD's lacking a label. If two different probes bearingdifferent labels are used, the flow cytometer can count GMD's bearingfirst label only, GMD's bearing second label only, GMD's bearing bothlabels, and GMD's bearing neither label. In methods employing largernumbers of probes, still further categories of GMD's can bedistinguished.

[0055] If a probe is directed to a particular sequence (e.g., a specificchromosomal defect), detection of GMD's hybridized to that probe signalsthat the defect is present in at least some of the biological entitiesbeing analyzed. If all GMD's bearing biological entities are labelledwith a second label, it is possible to determine a ratio of the GMD'shybridized to the specific probe with all GMD's encapsulating abiological entity. This ratio is the proportion of biological entitiesin a sample preparation bearing a defect. The methods are sufficientlysensitive to detect rare cells in larger populations, e.g., one cell in10, 100, 1000, 10,000, 100,000 or 1,000,000. This ratio can besignificant in determining the existence or prognosis of a disease. Forexample, if a sample of a cell is from a tissue suspected of beingsusceptible to cancer, the ratio of cells bearing a defect associatedwith cancer to the total number of cells in a sample is a measure of howfar the cancer has progressed.

[0056] In some methods, GMD's encapsulating chromosomes are hybridizedwith two different probes which are complementary to segments normallyfound on different chromosomes but which are translocated into the samechromosome in cancerous cells. In this situation, the ratio of GMD'sbinding to both probes relative to the GMD's binding to one probe or theother but not both, gives the ratio of cancerous to normal cells in apopulation.

[0057] Optionally, flow cytometry can be followed by FACS sorting tomake different classes of GMD's available for further analysis, such asmicroscopy, or chromosome preparation. Alternatively, gel microdropletscan be labelled with magnetic particles and subjected to magneticseparation (MACS). Magnetic particles can be directly attached tohybridization probes or can be supplied in a form that they specificallybind to hybridization probes.

[0058] VIII. Visualization of Hybridized Chromosomes

[0059] Encapsulated biological entities can be visualized with orwithout prior flow cytometry and FACS separation to determine thelocation(s) at which probe has bound. Analysis after flow cytometry andFACS separation can be advantageous because at that stage one has arelatively pure population of biological entities that has hybridized toa given probe. Biological entities can be visualized by microscopy,digital image analyzing, scanning cytometry, photon counting or ccd.Visualization is useful for analyzing hybridization of chromosomepainting probes or reverse chromosome painting probes. Visualization isalso useful for determining chromosomal copy number within a cell, andhence the existence of chromosomal deletions or duplications. Forbiological entities containing multiple chromosomes (e.g., cells andnucleic) visualization can also be used to distinguish between twodifferent probes binding to separate chromosomes or to the samechromosome. As noted, such analysis is useful in identifying some formsof cancer.

[0060] Biological entities are preferably immobilized on microscopeslides or the like for visualization. For example, placing a smallquantity (10 μl) of GMDs dispersed in substrate on the glass slide witha cover slip sufficiently immobilizes GMDs permitting reliable detectionof emitted light.

[0061] Digital imaging has become an indispensable tool for biologicalresearch due to several advantages when compared to the human eye. Thehigher sensitivity imaging detector enables one to visualize very lowlight objects which are not detectable by the unaided human eye. Thespectrum sensitivity of the human eye is limited from 400 to 700 nm. Incontrast, the spectrum sensitivity range of imaging detectors is morebroad, and signals from the range of x-ray to infrared can be detected.

[0062] A charged-coupled device (CCD) camera providing exposures rangingfrom seconds to minutes and has advantages for detecting low lightlevels. These cameras are coupled to a microscope; then digital imagesare collected with the help of appropriate instrumentation. For lowlight applications, there are two types of CCD cameras available. Thefirst is the Intensified CCD (ICCD) camera which uses an ImageIntensifier and a CCD camera. The Image Intensifier enhances low lightimage and the intensified image is projected onto a CCD camera throughrelay optics, such as a relay lens, enabling one to visualize low lightimage undetectable using a CCD alone. The second is a Cooled CCD (CCCD)camera which uses a similar CCD chip for high light imaging. The CCCDreduces camera noise by cooling and slowly reading out the signal. Thereduction of noise enables one to visualize a low light image ordinarilyburied in the noise of a regular CCD camera.

[0063] IX. DNA Isolation

[0064] The gel environment preserves chromosomal DNA molecules in intactform. DNA in encapsulated chromosomes containing GMD's can be cleaved tofragments in situ with restriction enzymes. For large fragments, partialdigestion is preferred. DNA fragments can then be released from drops bydigesting the gel matrix. For example, if the matrix is agarose, the gelmatrix can be digested with the enzyme agarose. Large fragments are thencloned into vectors such as YACs, BACs or PACs. Libraries from purifiedhuman chromosomes preparable by the above methods are useful forsequencing or mapping the human genome and for positional cloning. Puresorted chromosomes are also useful as a source of chromosome paintingand reverse chromosome painting probes. Such probes can be prepared byamplification of chromosomal DNA using degenerate primers.

[0065] X. Storage of Encapsulated Biological Entities

[0066] Encapsulated biological entities such as chromosomes can bestored for an hour, a day, a week, a month, six months, a year, twoyears or five years or more without visible degradation of nucleicacids. Stored chromosomes can eventually be used for isolating specificgenes, for preparing PCR probes, and for generating high qualitystarting material for DNA sequencing, or for clinical analysis.

[0067] XI. Applications

[0068] The above methods can be applied to diagnosing the presence,susceptibility, or prognosis of diseases associated with geneticdefects. The methods are particularly useful in diagnosing andmonitoring diseases due to genetic defects that are only present in asubpopulation of cells, such as defects arising from somatic mutation.Examples of such diseases associated with genetic defects includeautoimmune diseases, inflammation, cancer, diseases of the nervoussystem, and hypertension. Some examples of autoimmune diseases includerheumatoid arthritis, multiple sclerosis, diabetes (insulin-dependentand non-independent), systemic lupus erythematosus and Graves disease.Some examples of cancers include cancers of the bladder, brain, breast,colon, esophagus, kidney, leukemia, liver, lung, oral cavity, ovary,pancreas, prostate, skin, stomach and uterus.

[0069] Many cancers arise due to mutations in rare subpopulations ofcells. Cancers develop and progress through the accumulation of geneticabnormalities at critical loci (34, 35). These abnormalities may involvealterations of one or a few bases of DNA, deletions ranging fromsub-microscopic to whole chromosomes, duplications or higher-levelamplifications of chromosomal regions, or rearrangements producingabnormal juxtapositions of DNA sequences (36). In some case, such as theabl-bcr fusion on the original Philadelphia chromosome, specifictranslocations are associated with activation or modification of humanproto-oncogenes (37, 38). Similarly, Ewing's sarcoma is associated witha translocation involving the EWS gene on chromosome 22. Determinationof the proportion of cancerous cells in a tumor allows grading of thetumor for improved diagnosis, prognosis and treatment planning. Theratio can also be valuable in evaluating both the adequacy of surgicalmargins and the presence of microscopic metastases in bone marrow orother sites.

[0070] The methods are also useful for diagnosing the presence of latentviruses. For example, some viruses, such as Herpes viruses andretroviruses, integrate into genomic DNA of some cells within the bodyand remain dormant until activated. Analysis of a tissue sample from apatient having or suspected of being infected with such a virus canidentify the percentage of cells infected with the virus and the copynumber of the virus in different cells. Such information is useful indiagnosing the presence of virus, the severity of infection and therecommended course of treatment. Presence of a virus can be detectedusing a probe designed to hybridize either to viral genomic nucleic acidor to viral mRNA, or both. The methods can similarly be used todetermine copy number of viral mRNA in cells from the patient. Suchinformation can be useful in monitoring the progress of disease, forexample, in response to treatment with a drug.

[0071] The methods are also useful for determining allelic frequenciesin a population, and correlating such frequencies with a phenotype. Forexample, cells taken from a population of individuals can be pooled, andscreened to determine the frequencies of different allelic forms of agene. If the population has a common phenotype (e.g., a disease), acorrelation can be performed to determine whether the presence of one ofthe allelic forms is statistically associated with the phenotype.

[0072] The methods are also useful for identifying cell types expressinga gene of interest. For example, if a new gene of unknown function hasbeen discovered, one can design a probe that is complementary to anexonic segment and optionally, to segments in successive exons. Hence,the probe can hybridize to mRNA expressed from the gene. A population ofcells is obtained from different tissues of an individual, and the cellsare screened for hybridization to the probe. Cells hybridizing to theprobe express the gene. The nature of cells expressing a gene providesvaluable information concerning the function of the gene.

[0073] Similar methods can be used to clone a cDNA if only a portion ofthe coding sequence is known. The portion of known sequence is used todesign a probe. A population of different cells is then encapsulated andhybridized with the probe. Cells are then separated according to extentof expression. Cells showing the highest level of expression provide asuitable source material from which to clone the cDNA.

[0074] The methods are also useful for comparing or monitoring theexpression of a given gene or gene(s) in different cell types. A probeis designed to hybridize to a mRNA transcript of each gene of interest.Optionally, different probes can bear different labels. Probes are thenhybridized with mRNA in a microdrop encapsulated cell population, whichtypically includes cells of different types. The extent of hybridizationof each cell with each probe is then determined. Optionally, cellshybridizing with a particular probe at significantly above or belowaverage levels are isolated and cell type determined, allowingcorrelation between cell type and expression level. Optionally,different cell types in a population can themselves be labelled withreagents that specifically bind to a particular cell type. For example,a particular cell type can be labelled using an antibody that binds to areceptor specific to the cell type. Microdrops are then analyzed forboth cell type labels and probe labels, thereby facilitating comparisonof expression levels of particular mRNA species between the cell typesthat have been specifically labelled.

[0075] The methods are also useful for preparing isolated chromosomes.As noted, isolated chromosomes are useful for e.g., positional cloningstudies and for preparing probes.

[0076] XII. Kits

[0077] The invention also includes kits for the practice of the methodsof the invention. The kits comprise equipment and/or reagent(s) formaking gel microdrops and optionally, probe(s) for performinghybridization to encapsulated nucleic acids. Examples of equipmentinclude a CellSys 100™ Microdrop Maker and components thereof, aninstrument providing a pulsating novel, such as an inkjet printer, and avortexer. Examples of reagents include chemicals for making a gel, suchas agarose or acrylamide, cross-linking reagents, denaturing agents, andhybridization buffer. The kits can also include label(s) and otherchemicals to amplify label signal. The kits usually include labelling orinstructions indicating the suitability of the kits for performinghybridization in gel drops and/or flow cytometrix analysis. The term“label” is used generically to encompass any written or recordedmaterial that is attached to, or otherwise accompanies the kit at anytime during its manufacture, transport, sale or use.

EXAMPLES

[0078] 1. Encapsulation, Hybridization and Screening of Chromosomes, andStability of Encapsulated Chromosomes

[0079] Materials and Methods

[0080] Cell Lines and Culture Conditions Human chronic myelogenousleukemia, K-562, human acute lymphocytic leukemia, REH (American TypeCulture Collection, Rockville, Md.), and mouse fibroblast, Mus SpretusC1-SA (Los Alamos National Laboratory, Los Alamos, N. Mex.) cells weregrown in RPMI 1640 medium supplemented with 10% fetal bovine serum at37° C. in the presence of 5% CO₂. Normal human lymphoblast cells GM130(NIGMS Human Genetic Mutant Cell Repository, Coriell Institute forMedical Research, Camden, N.J.) were grown under identical conditions,except that RPMI 1640 medium was supplemented with 15% heat inactivatedfetal bovine serum.

[0081] Source of Plant Chromosomes Plant chromosomes from the field beanVicia faba, (from J. Dolezel, Institute of Experimental Botany, Olomouc,Czech Republic), were isolated from root meristems after cell cyclesynchronization with hydroxyurea and metaphase accumulation withamiprophos methyl (21).

[0082] Mitotic Cell Preparation Mouse C1-5A cells (an adherent cellline) undergoing logarithmic growth were treated with 0.2 μg/ml colcemid(Sigma, St. Louis, Mo.) for 12-15 hours. Mitotic cells, which becomeless adherent, were shaken-off and resuspended in hypotonic solution (55mM KCl). Human K-562 cells undergoing exponential growth were treatedwith 60 μM genistein (Sigma) for 24 hours to synchronize growth (23).Cells were then pelleted and washed once with Hank's balanced saltsolution (Sigma). Fresh media containing 0.1 μg/ml of colcemid was addedand cells were grown for an additional 24 hours. After counting, cellswere pelleted by centrifugation (100 g for 10 min at 4° C.) andresuspended in hypotonic solution (75 mM KCl). Human REH cells weregrown to stationary phase, to synchronize growth, then were left for 2days. Cells were harvested by low speed centrifugation and grown infresh media containing 0.1 μg/ml colcemid for 24 hours. After pelleting,cells were resuspended in hypotonic solution (75 mM KCl).

[0083] Chromosome Isolation Human and mouse chromosomes were isolatedusing the polyamine method (31,32), with minor modifications.Approximately 2×10⁷ cells were swelled in 10 ml of hypotonic solutioneither for 30 min (human REH) or for 1 hr (human K-562 and mouse C1-5Acells) at room temperature. After swelling, cells were pelleted by 5 mincentrifugation at 40 g, and gently resuspended in 0.1 ml of freshhypotonic solution. Two ml of chromosome isolation buffer (CIB; 15 mMTris-HCl, 80 mM KCl, 20 mM NaCl, 2 mM EDTA, 0.5 mM EGTA, 0.2 mMspermine, 0.5 mM spermidine, pH 7.2) supplemented with 0.1% (v/v)2-mercaptoethanol and 0.2% (v/v) Triton X-100 was added and solutionswere mixed by brief (10 sec) vortexing. Tubes were kept at 0° C. forapproximately 10 min. C1-5A cells were passed through a 25 gauge needleuntil chromosomes were released, as monitored by fluorescencemicroscopy. Nuclei and cellular fragments were removed by threesuccessive 5 min centrifugations at 40 g. The top two thirds of thesupernatant, after the final centrifugation, was kept at 4° C. for 16hr. The supernatant was then carefully decanted to avoid disturbing thesettled chromosomal pellet. Chromosomes were resuspended in 0.2 ml ofCIB and aggregates were pelleted by centrifugation for 5 min at 40 g.Chromosomes were either stored in CIB or immediately encapsulated inagarose gel microdrops (GMDs).

[0084] Chromosome Encapsulation The chromosome/agarose mixture (2.3%Type XII agarose; (Sigma) 0.1% Triton X-100 (Sigma) containing 10⁶chromosomes/0.55 ml) was prepared by melting the agarose in CIB at 100°C., cooling the agarose to 58° C., and adding 100 μl of chromosomesuspension and 50 μl of 11% (v/v) Triton X-100 (pre-warmed to 58° C.) to0.4 ml of melted agarose. The mixture was held at 58° C. for 5 min andadded dropwise to 15 ml of CelMiX™ 200 emulsion matrix (One CellSystems, Cambridge, Mass.) which was also pre-warmed to 58° C. GMDs weregenerated with a CellSys100™ Microdrop Maker (One Cell Systems,Cambridge, Mass.) equipped with a 1.6 cm blade using successive rotorspeeds of 1,500 rpm for 1 min at 20-25° C., 1,500 rpm for 1 min at 0°C., and 1,500 rpm for 5 min at 0° C. GMDs were separated from theemulsion matrix by centrifugation at 350 g for 10 min. The encapsulatedchromosome-containing pellet was washed twice with 13 ml of CIB,re-pelleted by centrifuging 5 min at 250 g, and stored at 4° C. in 10 mlof the same buffer.

[0085] Chromosome Staining Encapsulated chromosomes were stained withpropidium iodide (0.4 μg/ml) for microscopic examinations and with7-actinomycin (7AAD) at the same concentration, both from Sigma, forflow cytometry.

[0086] Hybridization Probes LSI™ 22q (bcr-locus specific, Vysis, DownersGrove, Ill.), which hybridizes to a 300 kb region of the bcr gene onchromosome 22, was used. This probe was directly labeled with SpectrumGreen T (fluorescein). After labeling, the probe size distributionranged from 50-500 nt. The DNA probe was denatured at 95° C. for 5 minbefore hybridization.

[0087] Microdrop In Situ Hybridization (MISH) of Human Chromosomes

[0088] Encapsulated chromosomal DNA was denatured in 0.1 N NaOH, 50%(v/v) ethanol for 1.5 min. GMDs were washed once in 0.5 M sodiumcarbonate, pH 10.2. After denaturation, the hydroxyl groups in theagarose microdrops were mildly crosslinked with 5 μM divinylsulfone byincubating 30 min at room temperature. Excess reactive groups fromdivinylsulfone were blocked with 2% (v/v) 2-mercaptoethanol for 30 minin 40 mM Tris-HCl, pH 8.0. GMDs were then washed with 2×SSC (0.15M NaCl,0.015M sodium citrate).

[0089] 100 μl (approximately 2×10⁵) of human chromosomes encapsulated inGMDs was hybridized with 2.0 μl of the denatured probes (probeconcentration was proprietary for the manufacturer) in a hybridizationmixture (2×SSC, 10% (v/v) dextran sulfate, 50 μg/ml salmon sperm ssDNAfor 16 hours at 68° C. After hybridization, non-specifically bound probewas removed by incubating GMDs with 1.0 ml 0.4×SSC at 72° C. for 5 min(LSI/22) and washing twice with 0.4×SSC at room temperature. Cot DNA wasadded to prevent non-specific hybridization to repetitive sequences.

[0090] Microscopy and Digital Image Analysis The integrity ofchromosomes after fluorescent staining or MISH was checked visuallyusing an Olympus BH-2 microscope (40×SPlan 0.4 objective or phasecontrast A40 PL 0.65 objective) equipped for epifluorescence withappropriate filters for fluorescein and 7AAD.

[0091] Flow Cytometric Analysis After staining or MISH, encapsulatedchromosomes were analyzed using a FACS Vantage flow cytometer (BectonDickinson Immunocytometry Systems, San Jose, Calif.) equipped with astandard 100 μm nozzle. LYSYS II Ver.2.0 software was used for dataanalysis. To remove large particles, the GMDs were sieved through a 53μm nylon mesh (Small Parts Inc., Miami Lakes, Fla.). For flow cytometryanalysis, a concentration of microdrops not exceeding 2×10⁵/ml was used.Forward and side scatter signals were analyzed on a log scale andexamined in scatter plot format, permitting identification of and gatingon GMDs. 7ADD fluorescence was used to identify GMDs containingencapsulated chromosomes. To identify 7ADD, an argon laser with a 356 nmspectral line was used. To identify chromosomes which hybridized to theSpectrum Green™ labeled probe, fluorescein (FITC) intensity wasmeasured. For measuring FITC fluorescence, an argon laser with a 488 nmspectral line was used.

[0092] Fluorescence Activated GMD Sorting Encapsulated chromosomes weresorted using a FACS Vantage fluorescence activated cell sorter (BectonDickinson) adapted with a macrosort option. The sheath pressure was setat 2 psi with a sample differential of 1 psi. A large diameter sampleline was used to avoid clogging. The GMD samples were sieved through a53 μm mesh before sorting. Sorting speed was in the range of 50FITC-labeled chromosomes/sec. Sorted GMD-encapsulated chromosomes werepelleted by low speed centrifugation and taken up with 20 μl of Antifadesolution (Oncor, Gaithersburg, Md.), diluted 1:1 with CIB and analyzedusing a fluorescence microscope.

[0093] Digestion of Denatured Encapsulated Chromosomal DNA with HindIIIEncapsulated chromosomes were denatured, as previously described, washedwith 40 mM Tris-HCl, pH 8.0 and subsequently digested with Proteinase K(2 mg/ml) supplemented with lithium dodecyl sulfate (1%) in CIB for 12hours at 50° C. Chromosomes were then washed three times with a 200-foldexcess of CIB and two times with 20-fold excess of HindIII digestionbuffer (50 mM NaCl/10 mM Tris-HCl, pH 8.0/10 mM MgCl₂). Digestion ofencapsulated chromosomes was done using 20 units of HindIII (Gibco-BRL)in a reaction volume of 50 μl for 90 min at 37° C. Gel loading bufferwas added and samples were electrophoresed as described below.

[0094] Gel Electrophoresis of Chromosomal DNA Both free and encapsulatedchromosomes were treated with proteinase K and lithium dodecyl sulfateas described above for three hours at 50° C. Gel-loading buffer (6×,0.25% bromphenol blue, 0.25% xylene cyanol FF, 30% glycerol) was addedand samples were electrophoresed on 0.8% SeaKem Gold (FMC BioProducts,Rockland, Me.) agarose in TAE buffer (0.04 M Tris-acetate, 1 mM EDTA) at56 volts for 2-6 hours. Propidium iodide was present in the gel (0.5μg/ml) and in the electrophoresis buffer (0.05 μg/ml) duringelectrophoresis. 0.25 μg of lambda DNA or 1.0 μg φX174 DNA cut withHinFI (Gibco-BRL) was used as a standard.

[0095] Chromosome Storage and Recovery Free and encapsulated chromosomeswere kept at 4° C. for up to 6 months in CIB. Agarose (from Pseudomonasatlantica, Sigma) was used to digest GMDs to recover chromosomes ofinterest. Encapsulated chromosomes were pelleted, resuspended inphosphate buffered saline solution, pH 7.0 (Sigma), and agarose (30units per 1000 GMDs) was added. This suspension was incubated at 40° C.for two hours, treated with proteinase K as described above, andchromosomal DNA was analyzed by gel electrophoresis.

[0096] Results

[0097]FIG. 1 shows encapsulated and unencapsulated human, mouse, andplant chromosomes. Encapsulated chromosomes appear visually more compactthan unencapsulated chromosomes, with narrow centromeric regions andtightly bound chromatid extensions. The centromeric region ofencapsulated chromosomes appear physically unseparated, which would bean indication of chromatid loss. Interestingly, encapsulation of intactplant chromosomes, which are approximately 3 times larger than humanchromosomes, was also successfully performed using this procedure.

[0098] Stability of Encapsulated Chromosomes To assess the long termstability of encapsulated chromosomes, we examined DNA fragmentationusing gel electrophoresis. As depicted in FIG. 2, no DNA fragmentationwas found by electrophoretic analysis, even 6 months afterencapsulation. In contrast, one month after isolation, DNA fromunencapsulated human and mouse chromosomes has a smeared bandingpattern, indicative of DNA fragmentation due to nuclease cleavage. Thisresult indicates that encapsulated chromosomal DNA remains intact andcan be used for preparing chromosome-specific libraries.Intact-isolation and long term stability of high quality, high molecularweight DNA will be a major convenience for researchers and an innovationfor sample storage for clinical use.

[0099] In Situ Hybridization of Encapsulated Chromosomes An importantimprovement was development of a matrix crosslinking method whichallowed use of up to 50% formamide and temperatures as high as 95° C.,necessary for reproducible hybridizations. The agarose hydroxyl groupswere mildly crosslinked with divinyl sulfone. Although this procedurealso partially crosslinks chromosomal DNA to the agarose matrix, becausethis process is reversible at pH 10, DNA can be released from GMDs afterMISH, which is important for eventual construction ofchromosome-specific libraries.

[0100] Using a FACS Vantage both for flow cytometric analysis and cellsorting, dual-parameter dotplots were produced by plottingFITC-fluorescence, which was detecting probes hybridized to thechromosomes versus 7AAD fluorescence, which as a general DNA stain wasused to detect all chromosomes. FIG. 3A shows flow sorting of humanchromosome 22 following microdrops in situ hybridization using a bcrlocus specific probe. Chromosomes were counter-stained with7-amino-actino-mycin (Vysis, Downer's Grove, Ill.). Chromosomes werecounter-stained with 7AAD. R1 includes high FITC, low 7AAD fluorescencespecific for gel microdrops containing chromosome 22. The scattergramalso display empty GMDs, encapsulated chromosomes, and noise. FIG. 3Bshows sorted chromosomes visualized using fluorescent microscopy. Thehybridization signal is depicted in green (FITC) color. Hybridizationsignals were amplified using the TSA method, which provides a 10-100increase in signal intensity.

[0101]FIG. 4 shows purity of chromosome 22 after sorting measure by thepolymerase chain reaction using primer sets specific from chromosomes10, 21 or 22. Amplification results for each primer set were comparedusing unsorted and sorted chromosome populations. Unsorted chromosomesgenerated specific amplicons for each primer (lanes 3-5), Sortedchromosome 22 showed no contamination with chromosome 10 (lane 8 vs. 60and were less than 1% contaminated with chromosome 21 (lane 8 vs. lane7).

[0102] Recovery of Encapsulated Chromosomes We determined that afterrecovering chromosomes from agarose microdrops, DNA was not fragmented,even 6 months after encapsulation. We also tested the integrity of DNAafter digesting microdrops with agarase and found that theelectrophoretic pattern was the same as that of untreated encapsulatedchromosomes used as controls. These observations show that after releasefrom gel microdrops, encapsulated chromosomes will be a convenientsource of high quality DNA for molecular genetics studies.

[0103] Digestion of Denatured Encapsulated Chromosomes A major concernfor eventual use of sorted chromosomes to construct chromosome-specificlibraries was that digestion of chromosomal DNA with restrictionenzymes, which is necessary for cloning large DNA fragments (>100 kb)into BAC libraries, would be impossible because denaturation woulddestroy the DNA secondary structure. To address this concern, we testedthe hypothesis that brief alkaline use would only partially denature theDNA and that subsequent digestion with proteinase K at 50° C. in lowsalt buffer overnight would restore double strandedness, thus makingspecific enzyme digestion possible. Human chromosomes isolated from theGM 130 cell line were digested with proteinase K and cleaved withHindIII. The electrophoretic result depicted in FIG. 5 shows that therestriction endonuclease HindIII cleaved restored DNA and generatedtypical sized fragments, demonstrating that MISH treated chromosomes canbe used for library construction.

Example 2 Construction of Chromosome-Specific BAC Libraries

[0104] A major unrealized goal of fluorescence in situ hybridizationassays has been the use of flow cytometry to isolate specificchromosomes for library construction. Prior to development of the MISHmethod, after hybridization, only a small fraction of chromosomes remainintact and free in suspension. Without the protection gained using theagarose microspheres, most chromosomes are clumped or fragmented, makingthem largely unsuitable for flow cytometric analysis (27). We have shownnot only that we can hybridize and flow sort encapsulated chromosomes,but also that alkaline-denatured chromosomes can be digested withrestriction endonucleases. As a result of this finding, we proposeconstruction of chromosome-specific libraries. Human chromosome 21 waschosen because it is often difficult to identify and sort usingconventional dual fluorescent staining since it is small andindistinguishable in the presence of cellular debris (29). The secondBAC library will be constructed using human chromosomes 9, which belongsto the group of chromosomes not resolvable by conventional dualfluorescent staining because of its similarity in size to several otherchromosomes (13). Chromosome libraries are constructed using the stepsbelow.

Sort MISH-labeled chromosomes ↓ Remove hybridized probes ↓ Digest sortedchromosomes with proteinase K in low salt buffer ↓ Digest chromosomalDNA with restriction enzymes ↓ Ligate DNA fragments into BAC vector ↓Electroporate ligated product into E. coli host Screen transformants forchromosome-specific inserts Example 3 Use of MISH-Sorted Chromosomes forReverse Chromosome Painting

[0105] The development of in situ hybridizations with flow sortedchromosome libraries (25,26), combined with non-isotopic signaldetection (30), has become a powerful approach for rapidly analyzinghuman chromosomal aberrations, such as aneuploidy and translocations.These techniques, termed chromosome painting, are becoming widely usedin clinical cytogenetics. However, small rearrangements, additions, ordeletions are not detectable using conventional chromosome painting, butthese aberrations are detectable by reverse chromosome painting, whichis performed using a probe prepared from aberrant chromosomes. A methodof using MISH-sorted chromosomes is depicted below.

Sort MISH-labeled aberrant chromosomes Deproteinize sorted chromosomes ↓First round of chromosomal DNA PCR amplification ↓ Biotin-label probeproduction by a second round of PCR amplification ↓ Use probe in FISHpainting of normal chromosome metaphase spreads Example 4 Detection ofthe Philadelphia Chromosome

[0106] The Philadelphia chromosome is a shortened chromosome 22 thatresults from a balanced translocation between chromosomes 9 and 22 withthe translocation breakpoints at 9q34 and 22q11. As a result of thistranslocation, most of the abl oncogene, located on chromosome 9, isjuxtaposed to part of the bcr gene, located on chromosome 22, creating anew bcr-abl gene fusion (40, 51, 52, 53, 54). This gene fusion encodesan abnormal protein with strong tyrosine kinase activity compared withthe weak tyrosine kinase activity of the normal abl protein. Theabnormal tyrosine kinase produced from bcr-abl causes increased cellproliferation and contributes to leukemogenesis by unknown cellularpathways.

[0107] Translocations such as bcr/abl are currently detected bycytogenetic examination of metaphase chromosome preparations preparedfrom bone marrow cultures. Although this method is adequate to detectmost chronic myelogenous leukemia cases (CML) in which Ph¹-positivechromosomal translocations are visible after banding due to the sizedifference in metaphase spreads, in acute lymphoblastic leukemia (ALL),cytogenetic examination is successful in only 65-80% of the cases,depending on the experience of each laboratory (55). The lower yield inALL is the result of the following factors. First, many ALL patientshave inaspirable bone marrows, so no cells are available. Second,lymphoblasts can be difficult to culture, so there is little enrichmentof leukemia cells. And third, the percentage of Ph¹ carrying cells in asample may be as low as 30%, in contrast with CML in which virtually100% of the cancerous cells in the sample carry Ph¹. In cases wherechromosomal rearrangements are not visible in classical metaphasespreads, which can occur in Ph¹-positive chromosomes, both in CML or ALLleukemias, fluorescence in situ hybridization, Southern blot (DNA)analysis, or polymerase chain reaction (PCR) analysis must be performedto detect rearrangements (55).

[0108] In CML, Southern blot analysis has been useful in detectingevidence of Ph¹ chromosome in patients in whom the cytogenetics arenegative despite a clinical presentation of CML (53). This approach isonly of limited utility in ALL because only 25-50% of Ph¹ chromosomepositive ALL patients have M-bcr (Major breakpoin't rearrangements).Nearly all M-bcr negative patients have translocation breakpoints inm-bcr (minor breakpoint rearrangements) anywhere in the first intron,which is 70-kb in size, requiring performance of an impractical numberof Southern blots in order to adequately investigate this entire region.

[0109] The chromosomal breakpoints in CML and acute leukemias may occurover large DNA sequences: 5.8 kb for the bcr region in CML and over 90kb for the first intron of the bcr 1 gene in acute leukemias. Therefore,PCR amplification of the fusion bcr/abl gene sequences cannot beperformed on DNA from patient specimens. All PCR based methods aredesigned to amplify and detect the abnormal fusion of mRNA (RT PCR). Thedesign of these methods takes advantage of the fact that mRNA sequencesare much shorter, lacking intron sequences. Assuming primers can bedesigned to amplify short stretches of mRNA specific for particulartranslocation (currently available for Ph¹ fused mRNA), amplified targetcan be detected after hybridization with specific probes followed by gelelectrophoresis and Northern blotting. The size of bcr/abl fused mRNAfor different patients varies within certain ranges for CML and acuteleukemia breakpoints, but the same size fusion mRNA is characteristicfor each malignant clone and can be monitored for each patient over theclinical course of the leukemia.

[0110] An advantage of using mRNA as a target for PCR amplification isthat mitotic cells are unnecessary. The practical limit of sensitivityof detection is approximately 1 malignant per 10,000 non-malignantcells. While the exquisite sensitivity of PCR could be advantageous inmolecular diagnostic testing, its use is troublesome in the clinicallaboratory. Contamination with minute amounts of amplified DNA and/orRNA from patient samples or cell line controls, even in the range of 1to 10 copies, may generate false-positive results. Aerosols andcarryover are the main sources of contamination. A potential problemwith monitoring the presence of the Ph¹ chromosome after chemotherapy orbone marrow transplant by RT PCR is the presence of residual deadleukemia cells with intact mRNA complicating therapeutic assessment.

[0111] The MISH technique combined with flow cytometric analysis makes asignificant contribution to analysis of translocations. Becausemetaphase chromosomes are required, only live leukemia cells contributePh¹ chromosomes for detection. As long as leukemia cells proliferate andreach mitosis, approximately 20 Ph¹ positive chromosomes in the presenceof 50,000 chromosomes, including those from Philadelphia-negative cells,can be detected in about 5 min. This corresponds to detection of 20cells with a single copy of Ph¹ chromosome, or 10 cells with twodefective copies, in an environment of approximately 1,000 cells whichare Ph¹ chromosome negative (2% leukemia cells present). To improvestatistical significance, analysis of 100,000 events can be performed inapproximately 10 minutes, provided that metaphase chromosomes areavailable from both cell types. In contrast, in current FISH procedureusing metaphase spreads obtained from bone marrow cultures which have amitotic index of 10%, one would have to find one Ph-positive spread inthe presence of 1,000 Ph-negative spreads, a labor intensive andimpractical approach by conventional microscopy.

[0112] Materials and Methods

[0113] Chromosome encapsulation and hybridization conditions were asdescribed above. The LSI™ bcr/abl DNA probe was used for in situhybridization. The probe was directly-labeled withSpectrumGreen™/SpectrumOrange™ which was designed to detecttranslocations between chromosome 9 and 22 (Vysis, Downers Grove, Ill.).This probe detects bcr/abl gene fusions, the molecular equivalent of thePhiladelphia chromosome (Ph¹), in both metaphase and interphase cells.It can be used to identify bcr/abl gene fusion involving either of thetwo breakpoint regions (M-bcr and m-bcr) in the bcr gene on chromosome22. The bcr/abl translocation probe is qualified for use on bothcultured lymphocytes and bone marrow cells. The LSI™ probe does notcontain repetitive sequences and is composed of an abl probe directlylabeled with SpectrumOrange fluorophore and a bcr probe directly labeledwith SpectrumGreen fluorophore. The abl probe begins between c-abl exons4 and 5 and continues for about 200 kb toward the telomere of chromosome9. The bcr probe begins either between bcr exons 13 and 14 (m-bcr) orbetween bcr exons 2 and 3 (M-bcr) and extends toward the centromereapproximately 300 kb crossing well beyond the m-bcr region. The probesize distribution is within a range of 50-500 nt (after labeling).

[0114] After hybridization, chromosomes containing M-bcr/abl gene fusionin Chronic Myelogenous Leukemia (CML) and in Acute LymphoblasticLeukemia (ALL) can be expected to contain fused orange and green signalsin translocated chromosome 22, which are sometimes perceived as yellow.Normal chromosomes 22 should display green signal and normal chromosomes9 should display orange signal. Hybridized chromosomes that have them-bcr/abl gene fusion in ALL should contain fused green/orange signal inchromosome 22 and faint green signal not fused with orange signal onchromosome 9 (from the chromosome 22 region between m-bcr and M-bcr thatis translocated to chromosome 9).

[0115] Results

[0116] Chromosome Isolation To increase the yield of human mitoticcells, K-562 cells were synchronized by a novel method using genistein,an isoflavone which blocks the cell cycle at G₂/M. An importantadvantage of genistein for cell cycle synchronization was that it didnot appear to penetrate cells and was easily eliminated by washing.After synchronization, growth of K-562 cells was not significantlyaffected by the presence of colcemid for up to 24 hours, and a highpercentage (75%) of cells, therefore, reached mitosis. Use of genisteinyielded a ready supply of millions of chromosomes facilitating extensiveexperimentation with encapsulation and fluorescent in situ hybridizationconditions. The method can also be used to isolate chromosomes for otherpurposes.

[0117] In FIG. 6, detection of translocated chromosome 22 leftchromosome 9 middle, and translocation-free chromosome 22 right by MISHare shown. Translocated chromosome 22 is identifiable by the presence ofboth green and red colors on a background of a blue colored wholechromosome. Fused red and green signals may be perceived as yellow(bottom left of FIG. 6). This yellow color also represents fused bcr/ablgene (translocated chromosome 22). Translocation-free chromosome 22 isdetectable by the presence of single green color and chromosome 9 by thepresence of single red color. Digital images created by a CELLscandigital image system with Exhaustive Photon Reassignment (EPR), whichwas available through collaboration, are presented here, but afluorescence microscope equipped with a triple bandpass filter for DAPI,fluorescein and rhodamine was adequate to identify translocations inPhiladelphia¹ chromosome.

Example 6 Analyzing Encapsulated Human Nuclei Using Microscopy and FlowCytometry

[0118] Analyzing MISH signals in nuclei provides improved contrastrelative to MISH detection in chromosomes. Moreover, MISH analysis ofnuclei can be performed in cases where cell proliferation is difficultor impossible. The applications are numerous and include detection of:translocations, deletions, monosomies or trisomies for diagnostic andprognostic analysis of aberrations. This method can also be used inresearch applications including gene amplifications and gene mapping. Inthis example, we use human nuclei with deleted chromosome 3 as a modelsystem. This genetic defect presents numerous clinical symptoms inaffected people including mental retardation and multiple congenitalanomalies.

[0119] Isolation of Nuclei

[0120] Nuclei are isolated from human lymphoblastoid cell line GM11428(NIGMS Human Genetic Mutant Cell Repository, NIH, Bethesda, Md.). HL-60cells are a source of normal nuclei (control) with no deletions in anychromosome. Cells in logarithmic phase of growth are collected andswelled in hypotonic solution (75 mM KCl) for 1 hour. Cells are thenpelleted by low speed centrifugation and resuspended in2-mercaptoethanol-free CIB buffer (1 million cells/ml). Triton X-100 isthen added to obtain final concentration of 0.2%. Nuclei released fromcells are kept in this solution for 1 hour at 4° C., then pelleted bycentrifugation at 100 x g and resuspended in original cell volume of thesame buffer and re pelleted. Finally, nuclei are resuspended at aconcentration of 10 million nuclei per ml of 2-mercaptoethanol-free CIBbuffer.

[0121] Encapsulation of Nuclei

[0122] Encapsulation of nuclei is performed essentially as describedpreviously for chromosome encapsulation. However, GMD size can beadjustment from 25-35 to 45-55 μm to account for the larger size ofnuclei by modification of blade speed during the emulsification process.

Example 7 Improved Method of Chromosome Isolation

[0123] Chromosomes can be synchronized in metaphase by culturing cellsfrom which chromosomes are to be isolated with the flavonoid genisteintogether with colcemid, which is commonly used to block the cell-cycleat metaphase. This procedure yields a mitotic index of about 75%: (i.e.,three out of four cells yielded metaphase chromosomes).

[0124] Chromosomes are released from cells by hypotonic treatment.Quantitative release of chromosomes requires at least some physicalshearing force, such as vortexing or passage through a gauge needle, andgenerate some cell debris. After chromosome release, nuclei are removedby low speed centrifugation to remove nuclei. To remove proteins,chromosome lysates are then dialyzed in a Slide-A-Lyzer™ (Pierce,Rockford, Ill.), which has a large pore membrane (100,000 molecularweight cut off), against a 100 fold volume of CIB (with two changes).Two alternatives for separating chromosomes from cellular debris can beemployed.

[0125] In the first approach, chromosomes are captured with magneticbeads conjugated to antibodies specific for the double stranded DNA ofhuman chromosomes. These antibodies are present in sera of patientssuffering from scleroderma (Chemicon International, Temecula, Calif.).The immunoglobulin fraction of the sera is isolated using proteinA-agarose (Pierce, Rockford, Ill.). The IgG fraction is thenbiotinylated with biotin-NHS ester (Molecular Probes, Eugene, Oreg.)using the manufacturer's procedure. Streptavidin magnetic microbeads(Miltenyi Biotec, Auburn, Calif.) are used to bind the biotinylated IgGfraction of the human sera from scleroderma patients. The microbeads arethen used to capture chromosomes. Chromosomes covered with magneticbeads are separated from non-chromosomal material by means of a strongfield MACS Separator (Miltenyi Biotec). This approach also allowsenrichment of occupied GMDs from the pool of mostly unoccupied GMDs.

[0126] In the second approach, cellular debris is separated fromchromosomes using antibodies specific for cytoskeletal proteinsconjugated to a solid support. In the chromosome release step, buffercontaining detergent (Triton X-100) is used to solubilize membranes. Butcytoskeletal proteins, such as fibronectin filaments, actin filaments,or intermediate microfilaments, are mostly present in an insoluble form.Although most microtubule proteins are solubilized by colcemidtreatment, some remain. Antibodies against cytoskeletal proteins(fibronectin, actin, vimentin), are available from Accurate Chemical &Scientific (Westbury, N.Y.), Biosource International (Camarillo, Calif.)and Cytoskeleton (Denver, Colo.). These antibodies are conjugatedthrough reductive amination to AminoLink Plus Coupling agarose gel(Pierce, Rockford, Ill.). 0.5 ml of antibody-derivatized gel (in aCompact Reaction Column, United States Biochemical, Cleveland, Ohio) isused to capture insoluble fragments of cellular debris by passingdialyzed and concentrated chromosome solution through a column.

Example 8 Detection with Chemiluminescent Substrates

[0127] Exothermic chemical reactions generally release energy in theform of vibrational or rotational excitation or heat. Inchemiluminescent reactions, however, the electronically excited state isreached by a chemical reaction and light rather than heat is generated.The energy-rich source in most chemiluminescent reactions is peroxide,hydroperoxide, 1,2 dioxetane, or dioxetane bonds. During the transitionof these excited intermediates to the electronic ground state, light isemitted in a process known as direct chemiluminescence. Novel acridaneor dioxetane chemiluminescent substrates, developed by Tropix, BioTecx,and collaborators at Lumigen allow detection of 10⁻¹⁹ moles ofhorseradish peroxidase or 10⁻²¹ moles of alkaline phosphatase (74-76), asubstantial improvement in sensitivity over fluorescence based detectionat 10⁻¹⁴ moles per liter.

[0128] Chemiluminescent substrates for alkaline phosphatase arecurrently all phenyl phosphate dioxetanes (PPD). PPD(4-methoxy-4-(3-phosphatephenyl)spiro[1,2dioxetane,-3,2′-adamantane]disodium salt) or CSPD® (chlorine derivative of PPD) are now widely usedin clinical immunossays and protein and nucleic acid detection testkits. PPD or CSPD® based substrates also contain fluorescent enhancerswhich promote more efficient generation of chemiluminescent lightthrough extended glow kinetics.

[0129] Other substrates such as PS-1, a substrate for HRP which has justbecome commercially available, exploits Lumigen's discovery that estersof N-alkylacridancarboxylic acid are efficiently oxidized by peroxidaseenzymes in the presence of hydrogen peroxide and a phenolic enhancer.The reaction, which requires only a minimal catalytic quantity of aperoxidase, converts the acridan compounds to the correspondingN-alkylacridinium ester. The PS-1 HRP substrate reaction produces anintense chemiluminescence, reaching a peak in approximately 10 minutes,with an extended decay over several hours. In a direct comparison withenhanced luminol based chemiluminescent reagents, previously the mostsensitive system for detecting HRP, PS-1 was shown to be 100 timesbrighter (74).

[0130] The current detection limit using fluorescence in situhybridization (FISH) and non-isotopically-labeled probes isapproximately 100 kb DNA target or 10-30 copies of mRNA (77-79).Although slightly stronger signals can be obtained using probes labeledwith radioactive isotopes, unacceptably long exposure times of days toweeks are required to detect approximately the same length DNA targets.Furthermore, health and disposal concerns make this technologyunsuitable for most laboratories.

[0131] In situ hybridization techniques have dramatically improved inrecent years both in terms of safety and sensitivity, primarily due touse of enzymes instead of isotopes as reporter molecules. Due to highreaction turnover, horseradish peroxidase (HRP) or alkaline phosphatase(AP) are the most frequently used reporter enzymes. Using reporterenzymes, localization of hybridization signals can be performed eitherwith calorimetric immunohistochemistry methods, which are lesssensitive, or with fluorescent methods, which increase sensitivity10-100 fold, in comparison to the non-enzymatic methods (80).

[0132] The highest detection limits for both reporter enzymes areobtained, however, using chemiluminescent substrates which facilitatethe emmission of light triggered by an enzymatic reaction. Approximatedetection limits of several approaches are shown below. Detection MethodDetection Limit^(a) Base Pairs^(b) Color 10⁻¹⁰-10⁻¹² 200-400Fluorescence 10⁻¹³-10⁻¹⁵ 100-200 Radioisotopes 10⁻²⁰  10-100 PCR 10⁻²²0.4-10  Chemiluminescence 10⁻¹⁹-10⁻²¹  10-100 Microdrop CL- 10⁻²¹  10-100^(c) moles, kilobases, per each GMD

[0133] Tyramide Signal Amplification

[0134] The recently available Tyramide Signal Amplification (TSA) system(NEN Life Sciences Products, Boston, Mass.) designed for fluorescence insitu hybridization was used in order to compare amplified fluorescencesignal with chemiluminescence. TSA technology (80) uses horseradishperoxidase (HRP) to catalyze deposition of fluorophore or biotin labeledtyramide near the site of the hybridized probe, proximal to the enzyme.HRP can deposit 10²-10³ tyramide molecules in 10 min, resulting inpowerful signal amplification. In addition, since tyramide can belabeled both with fluorophores (TSA-Direct) and biotin (TSA-Indirect),both fluorescent or chemiluminescent signal can be detected.

[0135] For fluorescent measurements after MISH, GMDs were incubated inTNB blocking buffer (0.1M Tris-HCl, pH 7.5, 0.15M NaCl, 0.5% BlockingReagent, NEN) with diluted conjugates of HRP with streptavidin forbiotin labeled probes, or anti-fluorescein for fluorescein labeledprobes (both from NEN) for 30 min. After three subsequent washes withTNT (0.1M Tris-HCl, pH 7.5, 0.15M NaCl, 0.05% TWEEN 20), GMDs wereincubated with 300 μl of diluted fluorescein-tyramide for 10 min.Unreacted tyramide was removed by washing GMDs twice with TNT. MISHsignals were then visualized using fluorescence microscopy.

[0136] Chemiluminescent Detection of Hybridized Probes

[0137] GMDs hybridized with biotin labeled probes were incubated for 30min in TNB blocking buffer containing conjugate of eitherstreptavidin-HRP (NEN) or HRP (Sigma) at dilutions of 1:100. GMDshybridized with fluorescein-labeled probes were incubated with theconjugate of anti-fluorescein-HRP (NEN) diluted 1:100. In some signalamplification experiments, prior to chemiluminescent measurements,tyramide labeled with biotin was subsequently bound to streptavidinconjugate of either HRP or AP. Unreacted conjugates were removed bythree successive washes with TNT. GMDs were than pelleted by low speedcentrifugation and resuspended in 0.05 ml 0.4×SSC. Aliquots diluted in aratio of 1:5 with the appropriate chemiluminescent substrate were thanexamined either using a microscope equipped with a photon countingdevice or a luminometer.

[0138] For horseradish peroxidase detection, three Luminol-based and oneacridinium-based substrates were used. Two luminol-based substrates,LumiGLO™ and BM Chemiluminescence ELISA Substrate, were obtained fromKirkegaard and Perry LaboraLories (KPL Inc., Gaithersburg, Md.) andBoehringer Mannheim Corp. (Indianapolis, Ind.), respectively. A thirdluminol-based substrate, NF-1, which does not require phenolicenhancers, was recently developed by BioTecx, Inc. (Houston, Tex.) andavailable for experimentation. The acridinium-based substrate, PS-1, wasobtained from Lumigen, Inc. (Southfield, Mich.).

[0139] For alkaline phosphatase detection, two adamantyl 1,2-dioxetanearyl phosphate (PPD)-based substrates were used. Lumi-Phos 530, obtainedfrom Lumigen, is a premixed formulation containing phenyl phosphatedioxetane, MgCl₂₁ cetyltrimethylammonium bromide, and an enhancer. CSPD,a derivative of PPD mixed with the Emerald II enhancer, was obtainedfrom Tropix, Inc. (Bedford, Mass.).

[0140] GMDs were incubated in substrate solutions for 5-10 min at roomtemperature for HRP or 10-15 min at 37° C. for AP. Light emitted fromGMDs soaked in chemiluminescent substrates was measured using anOPTOCOMP® I luminometer (MGM Instruments, Hamden, Conn.).

[0141] Derivatization of GMDs with HRP and AP

[0142] GMDs were formed from biotinylated agarose (FMC Bioproducts,Rockland, Me.) using standard emulsification procedures and sievedsequentially through 62 and 45 pm nylon mesh (Small Parts, Miami Lakes,Fla.) to obtain uniform size microdrops. GMDs were then blocked with TNBblocking buffer for 15 min. Aliquots containing 5×10⁵ GMDs were reactedwith appropriate dilutions of streptavidin-HRP or streptavidin-AP (bothfrom Sigma) for 30 min. After binding, GMDs were washed 3 times with TNTwashing buffer.

[0143] Digital Image Microscopy

[0144] For imaging chemiluminescence, an Olympus BH-2 microscope,equipped with phase contrast objectives for visualizing GMDs, wasconnected to a photon counting device through a C-mount. A Hamamatsu(Bridgewater, N.J.) C2400-32 ICCD (intensified CCD) camera was used as aphoton counting device. The camera consists of an image intensifier anda CCD camera coupled with a relay lens and a control unit (Hamamatsu IIcontroller, model M4314). In this configuration, the CCD cameraeffectively visualizes 756×485 pixels (pixel size 8.4×9.8 μm). Imageswere created and modified using the Hamamatsu ARGUS 20 Image Processor,which permits real time image observation.

[0145] For fluorescence imaging, the microscope was connected to aHamamatsu C5985 cooled CCD camera and images, were created using aHamamatsu controller and an Argus 20 Image Processor. The CCD camera wascooled to 20° C. below the ambient temperature with a built in Peltiereffect device. Both chemiluminescent and fluorescent images wereanalyzed and superimposed or pseudocolored using Adobe® Photoshop® 4.0software (Adobe Systems, San Jose, Calif.) running on Windows® 95operating system.

[0146] Results

[0147] This research has demonstrated the feasibility of detecting atleast 2-10 kb DNA sequences located on single copy genes using probeshybridized to encapsulated nuclei and then imaged using microscopy and aCCD camera. A 2.2 kb DNA sequence of a single copy gene located on humanchromosome Y can be detected using a chemiluminescent substrate for HRP.This sequence was not detectable using fluorescently labeled probes.

[0148] We evaluated a variety of substrates for both HRP and AP reporterenzymes. Although the lowest detection limit for HRP, measured in aluminometer, was obtained using acridinium-based substrate, low lightlevel required for microscopic visualization destroyed the substrate.Two PPD (phenyl phosphate dioxetane) based substrates for AP were alsounaffected by red light required to focus specimens for microscopy.

[0149] To detect low chemiluminescent light levels we used several CCDcameras, including a Hamamatsu Peltier effect-cooled CCD and aPhotometrics CH250 cooled to −40° C. Low light levels generated fromprobe hybridizing to single copy gene sequences, which had approximately4×10²-10³ molecules of reporter enzyme, were best detected using photoncounting devices. By comparison, about 10⁵ reporter molecules of enzymebound to agarose GMDs were needed for detection using a cooled CCDcamera, such as the Photometrics CH250 (see Table 1 below). TABLE 1 CCDcamera detection limits for reporter enzyme molecules conjugated tostreptavidin. Reporter enzyme molecules CCD Camera detected/GMDHamamatsu C5985 CCCD >10⁵   Photometrics CH250 CCCD 10⁵ HamamatsuC2400-32 ICCD 10³

Example 9 RNA detection in Encapsulated Cells

[0150] The ability rapidly to detect the presence of virus-specific orcancer-specific RNAs in individual cells present in low frequences hasapplications in disease diagnosis, monitoring and treatment as well asblood screening. Microdrop in situ hybridrization (MISII) isparticularly suited for these applications because it eliminates theneed for cell fixation and prevents cell clumping which makes thedetection of rare cells diffcult. This examples describes detecting bothcancer cells (positive for telomerase mRNA expression) and HIV-infectedcells by using a combination of MISH and flow cytometry.

[0151] Materials and Methods

[0152] 1. Cell Lines and Culture Conditions

[0153] Human promyelocytic leukemia HL-60 cells leukemia were purchasedfrom American Type Culture Collection (ATCC, Rockville, Md.). A3.01cells and HIV infected cells H9/HTLV-III NIH 1983 were obtained from theNIH AIDS Research and Reference Reagent Program, Rockville, Md. Allcells were cultured in RPMI 1640 medium supplemented with 10% fetalbovine serum (FBS) and were grown at 37° C. in the presence of 5% CO₂.

[0154] 2. Probes and Fluorescent Labeling

[0155] Oligonucleotide probes for detecting RNA labeled with fluoresceinat 5′ end were purchased from Oligoes Etc., Wilsonville, Oreg. Oligoslabeled with horseradish peroxidase were purchased from BiosorceInternational, Camarillo, Calif. Oligonucleotide probes for detection ofHIV RNA are derived from gag region of HIV genome. Sequences ofoligonucleotide probes are depicted below: HIV RNA detection H1 5′-(HRP)CCA TTC TGC AGC TTC CTC ATT GAT GGT CTC-3′ H2 5′-(HRP) CTT GTC TTA TGTCCA GAA TGC TGG TAG GGC-3′ Telomerase mRNA detection BF-1 5′FITC-CCA ACAAGA AAT CAT CCA CCA AAC GCA GGA GC 3′ BF-3 5′FITC-GAG GCT GTT CAC CTGCAA ATC CAG AAA CAG 3′ BF-4 5′FITC-GAA GGT TTT CGC GTG GGT GAG GTG AGGTG 3′

[0156] 3. Cell Encapsulation

[0157] Human cells harvested from various stages of growth were pelletedby low speed centrifugation, washed with Hanks Balanced Salt Solution(HBSS) containing 0.1% diethylpyrocarbonate (DEPC), and resuspended inthe same buffer at concentrations 20×10⁶ cells per ml. For encapsulationof cells, pluronic acid was used as a surfactant because it does notaffect cell membrane integrity during emulsification.

[0158] A cell-agarose mixture (2.3% Type XII agarose and 0.1% pluronicacid in IIBSS containing 2×10⁶ cells per 0.52 ml) was prepared bymelting 0.4 ml agarose in HBSS at 100° C., cooling the agarose to 57°C., and adding 100 μl of cell suspension and 20 μl of 10% pluronic acid.The mixture was held at 65° C. for 5 min and then quickly added dropwiseto 15 ml of CelMix 200 emulsion matrix (One Cell Systems, Cambridge,Mass.) pre-warmed to 6° C. Gel microdrops were created using aCellSys100 Microdrop Maker (One Cell Systems, Cambridge, Mass.) equippedwith a 1.6 cm blade using successive rotor speeds of 2,200 rpm for 1 minat 20-25° C., 2,200 rpm for 1 min at 0° C., and 1,200 rpm for 7 min at0° C. The GMDs were then separated from the emulsion matrix bycentrifugation at 400 x g for 7 min. The pellet containing encapsulatedcells was washed twice with DEPC treated HBSS and stored at 4° C. in thesame buffer and used within a week. However, encapsulated cells can bestored long-term (at least for a month) in 70% ethanol at −20° C.

[0159] 4. Microdrop In Situ Hybridization (MISH) of Encapsulated HumanCells

[0160] All solutions used for cellular RNA detection were prepared inDEPC treated water. 1 μl of DEPC (10% in 70% ethylalcohol) was added toapproximately 100,000 GMDs containing 10,000 cell-occupied GMDs in avolume of 50 pl. After a 15 min incubation at room temperature, an equalvolume of 2× hybridization buffer was added and the mixture wasincubated for a given time at temperatures ranging from 50 to 58° C. Forhybridization with the oligo probes, 2× hybridization solutioncontained, 1.2 M Tris-IIC1, pH 8.0, 0.1 mg/ml salmon sperm DNA, 0.1mg/ml E. coli tRNA, 100 units of placental RNAsc inhibitor/ml and 200 μlof Vanadyl Ribonucleoside Complex/ml (BRL, Bethesda, Md.). Before mixingwith encapsulated cells 10-20 picomoles/0.1 ml of oligonucleotide probeswere added to 2× hybridization buffer. After performing hybridization at55° C., GMDs were washed using wash buffer (WB, 0.05 M Tris-IIC1, pH0.0, 0.15 M NaCl, 0.2 mM EDTA, 0.1% Tween 20) at temperatures rangingfrom 20 to 55° C. Hybridization signals were amplified by a TyramideSignal Amplification (TSA, NEN™ Life Science Products, Boston, Mass.)either directly (with HRP labeled probes) or after binding withanti-fluoresecin-HRP (with fluorescein-labeled probes).

[0161] To detect HIV RNA with oligo probes directly labeled with HRP,encapsulated cells were first washed three times with HRP stabilizingbuffer (Biotecx, Cambridge, Mass.) before adding 2× hybridizationbuffer.

[0162] 5. Tyramide Signal Amplification

[0163] The Tyramide Signal Amplification (TSA) system (NEN Life SciencesProducts, Boston, Mass.) is designed for non-isotopic in situhybridization. TSA technology (37) uses horseradish peroxidase (HRP) tocatalyze deposition of tyramide labeled with fluorophores near the siteof the hybridized probe, proximal to the enzyme. HRP can deposit 10²-10³tyramide molecules in 10 min, resulting in powerful signalamplification. After MISH, GMDs were incubated in TNB blocking buffer(0.1M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.5% Blocking Reagent, NEN) with1:200 diluted conjugate of anti-fluorescein HRP (NEN) for 30 min. Afterthree subsequent washes with TNT (0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl,0.05% Tween 20), GMDs were incubated with 1:100 dilutedtyramide-fluorescein for 10 min. Unreacted tyramide was removed bywashing GMDs twice with TNT.

[0164] 6. Microscopy and Digital Image Analysis

[0165] The integrity of encapsulated cellular nucleic acids (both DNAand RNa after fluorescent staining with 0.2 μg/ml acridine orange) andMISH fluorescence signals were examined visually using an Olympus BH-2microscope (phase contrast 40 X SPlan 0.4 objective) equipped forepifluorescence with appropriate filters for DAPI, fluorescein orphycoerythrin. Digital images were taken with a cooled CCD camera (B/WHamamatsu C5985-02 with control unit) connected to the microscope.Digital images were processed with the assistance of computer software(Adobe Photoshop version 4.0, Adobe Systems, San Jose, Calif.).

[0166] 7. Flow Cytometric Analysis of Encapsulated Cells after MISH

[0167] Flow cytometric analysis after MISH was performed using an EPICSElite™ (Coulter Corporation, Miami, Fla.). 15 mW of a 488 nm line of anair cooled argon laser was used to excite the fluorescein in hybridizedprobes. The trigger parameter for collecting events was forward scatter(detecting encapsulated cells). 10,000 list mode events were collectedat a rate of approximately 600 occupied GMDs per second and analyzedusing elite 4.01 software.

[0168] Results

[0169]FIG. 7 shows flow cytometric detection of gag HIV RNA inencapsulated HIV infected cells after hybridization with two HRP-labeledoligo probes. A2.01 cells with the same lineage as H9 cells were used ascontrol cells. Both control and HIV infected cells were vultured for 4days. The peaks are color coded and represent the following: Red peak,A3.01 cells, probes not present (mean fluorescence 0.258); Blue peak, H9HTLVIII cells, probes not present (mean fluorescence=0.289); Yellowpeak, A3.01 cells, probes present (mean fluoresce ce=3.41); Green peak,H9HTI.VIII cells, probes present (mean fluorescence-19.2). Meanfluorescence of HIV infected and non-infected cells hybridized witholigo probes was used for the calculation of S/N value, which was foundto be 5. It has previously been estimated that H9/HTLV-III cells expressapproximately 250-500 copies of HIV-specific RNA. Detection of such alow copy RNA number indicates the sensitivity of the assay for detectionof HIV-infected cells in human blood.

[0170]FIG. 8 shows detection of telomerase mRNA in HL-60 (model cancercell line) and human PBMCs using fluorescein-labeled oligonucleotideprobes followed by TSA signal amplification. Hybridization conditionsand TSA amplification are described in Materials and Methods. Thehistograms are color coded as follows: red=HL-60 cells; black=humanPBMCs. Left peak represents unoccupiewd GMDs and right peak representsGMDs occupied with single cells. The relative mean fluoresence of theright peaks in 220 for HL-60 cells and 78 for human PBMCs (S/N-2.8).These results show the power of this methodology for detectingdifferential expression of mRNA in different cell types, andparticularly between normal and cancer cells.

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[0254] While the foregoing invention has been described in some detailfor purposes of clarity and understanding, it will be clear to oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention. All publications and patent documents cited inthis application are incorporated by reference in their entirety for allpurposes to the same extent as if each individual publication or patentdocument were so individually denoted.

What is claimed is:
 1. A method of nucleic acid analysis, comprisingforming a population of gel microdrops encapsulating a population ofbiological entities, each entity comprising a nucleic acid, whereby atleast some microdrops in the population each encapsulate a singleentity; contacting the population of gel microdrops with a probe underconditions whereby the probe specifically hybridizes to at least onecomplementary sequence in the nucleic acid in at least one gelmicrodrop; isolating or detecting the at least one gel microdrop.
 2. Themethod of claim 1, wherein the biological entities are selected from thegroup consisting of cells, viruses, nuclei and chromosomes.
 3. Themethod of claim 1, wherein the biological entities are not fixedchemically before the contacting step.
 4. The method of claim 1, furthercomprising amplifying the nucleic acids before the contacting step. 5.The method of claim 1, wherein the biological entities are chromosomes.6. The method of claim 1, wherein the population of gel microdrops isformed by forming a preparation of biological entities in a liquid gel,and dispersing the preparation into a hydrophobic solvent to form dropsencapsulating the entities.
 7. The method of claim 1, wherein thepopulations of gel microdrops is formed by forming a preparation ofbiologicial entities in a liquid gel and passing the preparation througha puslating orifice.
 8. The method of claim 7, wherein the pulsatingorifice is a component of an ink jet printer.
 9. The method of claim 1,wherein most drops contain zero chromosomes, and 1-30% of drops containa single chromosome.
 10. The method of claim 1, wherein the drops are2-200 μm in diameter.
 11. The method of claim 1, wherein the gel isselected from agarose, alginate, carrageenan, or polyacrylamide.
 12. Themethod of claim 1, further comprising digesting the gel at least oneisolated microdrop with agarase to isolate a nucleic acid within themicrodrop.
 13. The method of claim 1, further comprising denaturing thenucleic acid in the microdrops before the contacting step.
 14. Themethod of claim 1, wherein the gel is agarose and the method furthercomprising crosslinking hydroxyl groups in the agarose with each otherand with hydroxyl groups in the nucleic acid between the denaturationand contacting steps.
 15. The method of claim 14, wherein thehybridization is performed at a temperature of over 68° C. or in thepresence of a formamide concentration greater than 20%.
 16. The methodof claim 5, wherein the biological entities are are obtained from ahuman, nonhuman mammal, plant, bacterium, fungus, fish, or insect. 17.The method of claim 1, wherein the probe is labelled.
 18. The method ofclaim 17, wherein the microdrops further comprise a reagent thatamplifies a signal from the labelled probe.
 19. The method of claim 17,wherein the probe is labelled with an enzyme, and the reagent is asubstrate for the enzyme.
 20. The method of claim 17, wherein the probeis fluorescently labelled.
 21. The method of claim 1, wherein the probespecifically hybridizes to a subpopulation of the microdrops eachcontaining a nucleic acid bearing a complementary sequence to the probe.22. The method of claim 1, wherein the at least one gel microdrop isisolated by fluorescent activated cell sorting.
 23. The method of claim1, wherein the at least one gel microdrop is detected by flow cytometry,microscopy, digital image analyzing, scanning cytometry, photon countingor ccd.
 24. The method of claim 1, wherein the probe is a nucleic acid.25. The method of claim 24, wherein the probe is a locus-specific probe.26. The method of claim 5, wherein the probe comprises first and secondprobes respectively complementary to different chromosomes in a wildtypeindividual, whereby co-hybridization of the first and second probes tothe same chromosome indicates a chromosomal translocation in anindividual.
 27. The method of claim 1, wherein the probe hybridizes to asatellite DNA sequence, centromeric, a telomeric region or a repetitivesequence.
 28. The method of claim 1, wherein the probe is a chromosomespecific probe.
 29. The method of claim 5, wherein the population ofchromosomes are obtained from a single cell or a homogeneous cell linefrom a patient.
 30. The method of claim 1, further comprising labellingmicrodrops containing an entity with a second label without labellingempty microdrops with the second label.
 31. The method of claim 5,wherein the population of chromosomes is obtained from a population ofdifferent cells in a patient.
 32. The method of claim 31, furthercomprising determining the ratio of a subpopulation of microdropscontaining a chromosome hybridized to the probe to a supopulation ofmicrodrops containing a chromosome not hybridized to the probe.
 33. Themethod of claim 32, wherein the ratio is less than 1:10.
 34. The methodof claim 32, wherein the probe hybridizes to a nucleic acid segmentbearing a mutation and the ratio indicates the proportion of cells inthe population bearing the mutation.
 35. The method of claim 32, whereinthe mutation is a somatic mutation.
 36. The method of claim 32, whereinthe mutation is a germline mutation.
 37. The method of claim 1, furthercomprising contacting an isolated gel drop containing a nucleic acidwith a restriction enzyme, whereby the restriction enzyme cleaves thenucleic acid within the drop.
 38. The method of claim 5, furthercomprising preparing a single chromosomal fragment library from achromosome in an isolated gel microdrop.
 39. The method of claim 5,further comprising preparing probes from a single chromosome in anisolated gel microdrop.
 40. The method of claim 39, wherein the probesare chromosome painting probes.
 41. The method of claim 39, wherein theprobes are reverse chromosome painting probes.
 42. The method of claim1, further comprising storing a gel microdrop encapsulating a biologicalentity for at least one hour.
 43. The method of claim 42, wherein thebiological entity is stored before the contacting step.
 44. The methodof claim 42, wherein the biological entity is stored after the isolatingor detecting step.
 45. The method of claim 42, wherein the gel microdropis stored for at least six months.
 46. The method of claim 5, furthercomprising viewing the isolated gel microdrop under a microscope todetermine which regions of the chromosome have hybridized to the probe.47. The method of claim 1, further comprising contacting the at leastone gel drop containing a nucleic acid hybridized to the probe with alabel that binds to the probe.
 48. The method of claim 1, wherein thepopulation of chromosomes comprises at least 10,000 chromosomes.
 49. Themethod of claim 1, wherein the biological entities are cells and theprobe hybridizes to an RNA molecule with the cells.
 50. A method ofdiagnosing a disease due to a mutation, comprising: obtaining a sampleof cells from a patient; encapsulating a population of chromosomes fromthe sample in a population of microdrops; contacting the microdrops witha first probe that is complementary to a nucleic acid segment containingthe somatic mutation, and a second probe complementary to the chromosomein which the somatic mutation occurs at a site distal to the somaticmutation, whereby the first probe hybridizes to microdrops bearing thechromosome with a somatic mutation and the second probe hybridizes tomicrodrops bearing the chromosome irrespective whether the somaticmutation is present; determining the ratio of microdrops hybridizing tothe first probe and hybridizing to the second probe; diagnosing theexistence or prognosis of the disease from the ratio.
 51. The method ofclaim 50, wherein the disease is cancer.
 52. The method of claim 51,wherein the mutation occurs in a p53, BRCA-1, BRCA-2, ras orretinoblastoma gene.
 53. A method of chromosome analysis, comprisingforming a population of gel micropdrops encapsulating a population ofnucleic, whereby at least some microdrops in the population eachencapsulate a single nucleus; contacting the population of gelmicrodrops with a probe under conditions whereby the probe specificallyhybridizes to at least one complementary sequence in at least onechromosome in a nucleus of least one gel microdroplet; isolating ordetecting the at least one gel microdroplet.
 54. A method of isolatingchromosomes comprising: culturing a population of cells in genistein andcolcemid to synchronize chromosomes in metaphase; isolating chromosomesfrom the cells.
 55. A method of isolating chromosomes comprising: lysinga population of cells to form a lysate; treating the lysate with anantibody linked to a magnetic particles, wherein the antibodyspecifically binds to one or more chromosomes in the cells; isolatingmagnetic particles from the lysate.
 56. A method of chromosome analysis,comprising forming a population of gel micropdrops encapsulating apopulation of cells or nuclei, whereby at least some microdrops in thepopulation each encapsulate a single nucleus; contacting the populationof gel microdrops with a probe under conditions whereby the probespecifically hybridizes to at least one complementary sequence in atleast one nucleus in at least one gel microdrop; isolating or detectingthe at least one gel microdrop.
 57. The method of claim 56, wherein theprobe is labelled with an enzyme and the gel microdrops contain asubstrate for the enzyme.
 58. The method of claim 56, wherein gelmicrodrops are formed from a biotinylated gel, and the. substrate islinked to biotin via an avidin or streptavidin moiety.
 59. The method ofclaim 58, wherein the substrate is chemieluminescent.
 60. The method ofclaim 59, wherein the enzyme is horseradish peroxidase or alkalinephosphatase.
 61. The method of claim 56, wherein the detectingcomprising analyzing the at least one gel microdrop with a fluorescencemicroscope, a digital image analyser, a scanning cytometer, a photoncounting device or a ccd.
 62. The method of claim 61, wherein thedetecting indicates the distribution of the probe within the nucleus ofthe at least one microdrop.
 63. The method of claim 56, wherein thedetecting comprising placing a plurality of gel microdrops on amicroscope slip and detecting a hybridization signal using afluorescence microscope, a digital image analyser, a scanning cytometer,a photon counting device or a ccd.
 64. The method of claim 56, whereinthe probe hybridizes to a single copy genomic sequence shorter than 50kb.
 65. The method of claim 56, wherein the probe hybridizes to a singlecopy genomic sequence shorter than 10 kb.
 66. The method of claim 56,wherein the isolating or detecting is effected by flow cytometry,optionally with FACS, or MACS.
 67. A kit comprising a high meltingtemperature agarose, emulsification equipment, a label indicating how touse the kit for probe hybridization analysis.
 68. The kit of claim 67,further comprising at least one probe that hybridizes to a nucleic acid.